Tumor necrosis factor α (TNF-α or TNF-alpha) is a pleiotropic cytokine that is primarily produced by activated macrophages and lymphocytes; but is also expressed in endothelial cells and other cell types. TNF-α is a major mediator of inflammatory, immunological, and pathophysiological reactions. (Grell, M., et al., (1995) Cell, 83:793-802, incorporated entirely by reference). Two distinct forms of TNF exist, a 26 kDa membrane expressed form and the soluble 17 kDa cytokine which is derived from proteolytic cleavage of the 26 kDa form. The soluble TNF polypeptide is 157 amino acids long and is the primary biologically active molecule.
TNF-α exerts its biological effects through interaction with high-affinity cell surface receptors. Two distinct membrane TNF-α receptors have been cloned and characterized. These are a 55 kDa species, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R (Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840, incorporated entirely by reference). The two TNF receptors exhibit 28% similarity at the amino acid level. This is confined to the extracellular domain and consists of four repeating cysteine-rich motifs, each of approximately 40 amino acids. Each motif contains four to six cysteines in conserved positions. Dayhoff analysis shows the greatest intersubunit similarity among the first three repeats in each receptor. This characteristic structure is shared with a number of other receptors and cell surface molecules, which comprise the TNF-R/nerve growth factor receptor superfamily. TNF signaling is initiated by receptor clustering, either by the trivalent ligand TNF or by cross-linking monoclonal antibodies (Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627-1638, incorporated entirely by reference).
Crystallographic studies of TNF and the structurally related cytokine, lymphotoxin (LT) have shown that both cytokines exist as homotrimers, with subunits packed edge to edge in a threefold symmetry. Structurally, neither TNF or LT reflect the repeating pattern of the their receptors. Each monomer is cone shaped and contains two hydrophilic loops on opposite sides of the base of the cone. Recent crystal structure determination of a p55 soluble TNF-R/LT complex has confirmed the hypothesis that loops from adjacent monomers join together to form a groove between monomers and that TNF-R binds in these grooves. Random mutagenesis has been used to identify active sites in TNF-α responsible for the loss of cytotoxic activity (Van Ostade, X., et al., (1991) EMBO J., 10:827-836), incorporated entirely by reference. Human TNF muteins having higher binding affinity for human p75-TNF receptor than for human p55-TNF receptor have also been disclosed (U.S. Pat. No. 5,597,899 and Loetscher et al., J. Biol. Chem., 268(35) pp 263050-26357 (1993), both incorporated entirely by reference).
The different activities of soluble TNF (solTNF) and transmembrane TNG (tmTNF), mediated through discrete interactions with receptors TNFR1 and TNFR2, may account for contrasting beneficial and harmful roles reported for TNF in animal models and in human disease (Kollias, D. Kontoyiannis, Cytokine Growth Factor Rev. 13, 315 (2002); M. Grell et al., Cell 83, 793 (1995); M. Grell, H. Wajant, G. Zimmermann, P. Scheurich, Proc. Natl. Acad. Sci. U.S.A. 95, 570 (1998); C. O. Jacob, Immunol. Today 13, 122 (1992); R. N. Saha, K. Pahan, J. Neurochem. 86, 1057 (2003); and, M. H. Holtmann, M. F. Neurath, Curr. Mol. Med. 4, 439 (2004), all incorporated entirely by reference). For example, paracrine signaling by solTNF is associated with chronic inflammation, while juxtacrine signaling by tmTNF plays an essential role in resolving inflammation and maintaining immunity to pathogens (Holtmann & Neurath, supra; S. R. Ruuls et al., Immunity 15, 533 (2001); M. Canault et al., Atherosclerosis 172, 211 (2004); C. Mueller et al., J. Biol. Chem. 274, 38112 (1999); M. L. Olleros et al., J. Immunol. 168, 3394 (2002); and, M. Pasparakis, L. Alexopoulou, V. Episkopou, G. Kollias, J. Exp. Med. 184, 1397 (1996), all incorporated entirely by reference.) Excess soluble TNF levels are associated with numerous inflammatory and autoimmune diseases, and inactivation of TNF by injectable protein inhibitors reduces symptoms and blocks disease progression (B. B. Aggarwal, A. Samanta, M. Feldmann, in Cytokine Reference J. J. Oppenheim, M. Feldmann, Eds. (Academic Press, London, 2000) pp. 413-434, incorporated entirely by reference). The three FDA-approved TNF inhibitors include a TNFR2-IgG1 Fc decoy receptor (etanercept) and two neutralizing monoclonal antibodies, Remicade® (infliximab) and Humira® (adalimumab). Although effective anti-inflammatory agents, these immunosuppressive drugs can exacerbate demyelinating disease, induce lymphoma, reactivate latent tuberculosis, and increase the risk of sepsis and other infections, as indicated in their warning labels, (N. Scheinfeld, J. Dermatolog. Treat. 15, 280 (2004), incorporated entirely by reference). A possible explanation for the increased risk of infection comes from studies using TNF knockout and tmTNF knock-in mice, which demonstrate that tmTNF signaling is sufficient to maintain immunity to listerial and mycobacterial infection. In contrast, solTNF is a primary driver of inflammation. Decoy receptors and antibodies can bind to tmTNF, and that etanercept, infliximab, and adalimumab inhibit tmTNF in addition to solTNF (J. Gerspach et al., Microsc. Res. Tech. 50, 243 (2000); H. Mitoma, T. Horiuchi, H. Tsukamoto, Gastroenterology 126, 934 (2004); J. Agnholt, J. F. Dahlerup, K. Kaltoft, Cytokine 23, 76 (2003); B. Scallon et al., J. Pharmacol. Exp. Ther. 301, 418 (2002); C. Shen et al., Aliment. Pharmacol. Ther. 21, 251 (2005); and, H. Mitoma et al., Gastroenterology 128, 376 (2005), all incorporated entirely by reference). In view of the serious side effects of existing therapies, a therapeutic that is more potent and has a reduced side effect profile is still needed. The present invention shows that an anti-inflammatory agent that inhibits solTNF but spares tmTNF-mediated signaling will block inflammation yet preserve normal immunity to infectious agents.